The proposed project involves examining how the protein alpha-synuclein, which has been implicated in the pathology of both sporadic and inherited forms of Parkinson's Disease, affects the properties of the synaptic vesicle cycle in mammalian neurons. Our lab has previously found that overexpressing alpha-synuclein in mammalian neurons leads to an inhibition of synaptic vesicle exocytosis. The proposed research will attempt to answer a number of questions related to this phenotype. First, it is as of yet unclear whether the inhibition of exocytosis by alpha-synuclein represents a function that is normally performed by the protein or whether it is the result of toxicity gained through an increase in alpha-synuclein expression. To address this question, I am proposing to examine synaptic vesicle cycling in neurons harvested from mice that lack all endogenous forms of synuclein. If the phenotype in these neurons is the opposite of that in neurons overexpressing alpha-synuclein, namely if there is an increase in synaptic vesicle exocytosis, it would support the idea that modulating neurotransmitter release is indeed a normal physiological function of alpha-synuclein. This will also help in determining whether increased levels of normally-functioning alpha-synuclein may contribute to the pathogenesis of Parkinson's Disease. In addition, the proposal seeks to determine what region of the alpha-synuclein protein underlies its ability to inhibit synaptic vesicle exocytosis. Since the human form of alpha-synuclein seems to be more potent in its ability to inhibit exocytosis than the mouse form, I will focus my analysis on regions of the protein that are not conserved amongst mammalian species. Finally, the proposal will examine the possibility that alpha-synuclein acts indirectly to inhibit synaptic vesicle exocytosis by affecting another family of synaptic proteins, the synapsins. To address this possibility, I will overexpress alpha-synuclein in neurons taken from mice that lack all endogenous forms of the synapsins. If these neurons exhibit no deficiencies in synaptic vesicle exocytosis, it would give credence to the idea that alpha-synuclein acts by perturbing the normal function of the synapsins. Taken together, these studies will therefore help to gain insight into the physiology of alpha- synuclein and to understand how this protein contributes to the pathology of Parkinson's Disease. PUBLIC HEALTH RELEVANCE: Parkinson's Disease represents a substantial financial and emotional burden to the public. Understanding the physiology of genes linked to the inheritance of the disease represents a crucial first step towards developing novel therapeutic strategies that aim to alleviate this burden. The proposal seeks to understand the contributions of the protein alpha-synuclein to the etiology of Parkinson's Disease and is therefore directly related to the goal of treating or preventing Parkinson's Disease and consequent improvement of public health.